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Biologie

响应电离照射的肠上皮再生

Published: July 27, 2022
doi:
10.3791/64028
, , , ,
1Department of Medicine,Renaissance School of Medicine at Stony Brook University, 2Department of Physiology and Biophysics,Renaissance School of Medicine at Stony Brook University

Summary

胃肠道是放射治疗癌症治疗后对损伤最敏感的器官之一。它同时也是一个器官系统，在这种侮辱之后具有最高的再生能力。所提出的协议描述了一种研究肠上皮再生能力的有效方法。

Abstract

肠上皮由单层细胞组成，但包含多种类型的终末分化细胞，这些细胞是由位于肠隐窝底部的肠道干细胞的活跃增殖产生的。然而，在急性肠损伤事件期间，这些活跃的肠道干细胞会经历细胞死亡。伽马射线照射是一种广泛使用的结直肠癌治疗方法，虽然治疗有效，但具有耗尽活性干细胞库的副作用。事实上，患者在接受放疗时经常出现胃肠道辐射综合征，部分原因是活跃的干细胞耗竭。肠隐窝中活性肠道干细胞的丢失激活了通常静止储备的肠道干细胞库，并诱导分泌细胞和肠细胞前体细胞的去分化。如果没有这些细胞，肠上皮将缺乏从放疗和其他此类主要组织损伤中恢复的能力。谱系追踪技术的新进展允许跟踪再生过程中细胞的活化、分化和迁移，并已成功用于肠道研究。本研究旨在描述一种分析辐射损伤后小鼠肠上皮内细胞的方法。

Introduction

如果完全平放，人类的肠上皮将覆盖大约半个羽毛球场的表面1.相反，这种将人类与肠道内容物分开的单细胞层被压缩成一系列手指状的突起、绒毛和凹痕，这些隐窝使肠道的表面积最大化。上皮细胞沿隐窝绒毛轴分化。绒毛主要由吸收营养的小肠细胞、分泌粘液的高脚杯细胞和产生激素的小肠内分泌细胞组成，而隐窝主要由产生防御素的潘氏细胞、活性和储备干细胞以及祖细胞2，3，4，5组成。此外，这些细胞与底层间充质区室的基质和免疫细胞以及腔内微生物群的双向通信产生了维持肠道稳态的复杂相互作用网络，并且对损伤后的恢复至关重要6，7，8。

肠上皮是人体内自我更新最迅速的组织，周转率为2-6天9，10，11。在体内平衡期间，位于肠隐窝基部的活性干细胞（隐窝基柱状细胞）以表达富含亮氨酸的重复含 G 蛋白偶联受体 5 （LGR5） 为标志，快速分裂并提供分化为所有其他肠上皮谱系的祖细胞。然而，由于其高有丝分裂率，活性干细胞及其直系祖细胞对伽马辐射损伤特别敏感，并在照射后发生细胞凋亡5，12，13，14。在它们丢失时，肠隐窝内的储备干细胞和非干细胞（祖细胞亚群和一些终末分化细胞）经历激活并补充基底隐窝隔室，然后可以重建绒毛的细胞群，从而再生肠上皮15。使用谱系追踪技术，多个研究小组已经证明，储备（静止）干细胞能够在活性干细胞丢失时支持再生13，16，17，18，19，20，21，22。这些细胞的特征在于存在多梳复合蛋白 1 癌基因 （Bmi1）、小鼠端粒酶逆转录酶基因 （mTert）、啤酒花同源盒 （Hopx） 和富含亮氨酸的重复蛋白 1 基因 （Lrig1）。此外，已经表明非干细胞能够在受伤时补充肠道隐窝23，24，25，26，27，28，29，30，31。特别是，已经表明分泌细胞和肠细胞的祖细胞在损伤时经历去分化，恢复为干细胞样细胞，并支持肠上皮的再生。最近的研究已经确定了表达多种标记物的细胞，这些标记物具有在损伤时获得干样特征的能力（例如DLL +，ATOH1 +，PROX1 +，MIST1 +，DCLK1 +）32，33，34，35，36。令人惊讶的是，Yu等人表明，即使是成熟的潘氏细胞（LYZ+）也可以促进肠道再生37。此外，除了引起肠上皮细胞凋亡和破坏上皮屏障功能外，照射还会导致肠道菌群失调，免疫细胞活化和促炎反应的启动，以及间充质和基质细胞的活化38，39。

伽马辐射是癌症治疗中有价值的治疗工具，特别是对于结直肠肿瘤40。然而，照射通过诱导细胞损伤来显着影响肠道稳态，从而导致细胞凋亡。辐射暴露会导致多种扰动，从而减慢患者的康复速度，并以急性期粘膜损伤和炎症以及长期腹泻、尿失禁、出血和腹痛为特征。这一系列表现被称为胃肠道辐射毒性。此外，辐射诱导的透壁纤维化和/或血管硬化的进展可能仅在治疗后数年出现38，41。在损伤本身的同时，辐射在肠道细胞中诱导修复反应，激活负责启动和协调再生的信号通路42。放射诱发的小肠疾病可起源于向其他器官（如子宫颈、前列腺、胰腺、直肠）提供的盆腔或腹部放疗41，43，44，45，46。因此，肠道照射损伤是一个重要的临床问题，更好地了解由此产生的病理生理学可能会推动干预措施的发展，以减轻与放疗相关的胃肠道并发症。除了辐射之外，还有其他技术可以研究肠上皮的再生目的。已经开发了用于研究炎症和此后再生的转基因和化学小鼠模型47。葡聚糖硫酸钠（DSS）诱导肠道炎症，并导致与炎症性肠病相似的特征的发展48。DSS治疗与促癌化合物嘧啶甲烷（AOM）的组合可导致结肠炎相关癌症的发展48，49。缺血再灌注诱导损伤是另一种用于研究肠上皮再生电位的方法。这种技术需要经验和外科知识50.此外，上述技术引起的伤害类型与辐射不同，并可能导致不同再生机制的参与。此外，这些模型非常耗时，而辐射技术相当简短。最近，利用肠和结肠产生的肠和结肠样的体外方法已被与辐射损伤结合使用，以研究肠道再生的机制51，52。然而，这些技术并不能完全概括它们建模的器官53，54。

提出的方案包括对γ辐射损伤小鼠模型的描述，以及结合遗传模型，在他莫昔芬治疗后，允许追踪源自储备干细胞群的谱系（Bmi1-CreER;罗莎26eYFP）。该模型利用 12 Gy 全身照射，诱导足够显着的肠道损伤以激活储备干细胞，同时仍允许在受伤后 7 天内对肠道再生能力进行后续研究55。

Protocol

所有小鼠都被安置在石溪大学的实验动物资源部（DLAR）。石溪大学机构动物护理和使用委员会（IACUC）批准了所有涉及动物受试者的研究和程序。涉及动物受试者的实验严格按照批准的动物处理方案（IACUC #245094）进行。 注意：商业上获得小鼠品系B6;129-Bmi1 tm1（cre/ERT）Mrc/J（Bmi1-Cre ER）和B6.129X1-Gt（ROSA）26Sortm1（EYFP）Cos/J（Rosa26eYFP）（?…

Representative Results

将 12 Gy 全身照射 （TBI） 与小鼠遗传谱系追踪相结合，可以对肠道辐射损伤的后果进行彻底分析。首先， Bmi1-CreER;Rosa26eYFP小鼠接受单次他莫昔芬注射，该注射液在Bmi1 + 储备干细胞群中诱导增强的黄色荧光蛋白（EYFP）表达。注射他莫昔芬两天后，小鼠接受照射或假照射。安乐死前三小时，小鼠被注射EdU。安乐死后，收集小肠标本在照射后0 h、3 h、6 h、24 h、48 h、72 h?…

Discussion

该协议描述了一个稳健且可重复的辐射损伤模型。它允许精确分析损伤后7天内肠上皮的变化。重要的是，所选的时间点反映了损伤的关键阶段，其特征是肠道的明显改变（损伤、细胞凋亡、再生和正常化阶段）60。已经建立并仔细评估了这种照射模型，证明了一种合适的损伤表现，以模仿接受放射治疗的患者所经历的损伤表现61。超过一半的结直肠癌和妇科癌?…

Divulgations

The authors have nothing to disclose.

Acknowledgements

作者希望感谢石溪癌症中心组织学研究核心在组织标本制备方面的专家协助，以及石溪大学实验动物资源部在动物护理和处理方面的协助。这项工作得到了美国国立卫生研究院DK124342的资助，授予Agnieszka B. Bialkowska和DK052230授予Vincent W. Yang博士。

Materials

1 mL syringe BD 309659 –
16G Reusable Small Animal Feeding Needles: Straight VWR 20068-630 –
27G x 1/2" needle BD 305109 –
28G x 1/2" Monoject 1mL insulin syringe Covidien 1188128012 –
5-Ethynyl-2′-deoxyuridine (EdU) Santa Cruz Biotechnology sc284628A 10 mg/mL in sterile DMSO:water (1:4 v/v), aliquot and store in -20°C
Azer Scientific 10% Neutral Buffered Formalin Fisher Scientific 22-026-213 –
B6.129X1-Gt(ROSA)26Sortm1(EYFP)Cos/J The Jackson Laboratory Strain #:006148
B6;129-Bmi1tm1(cre/ERT)Mrc/J The Jackson Laboratory Strain #:010531
Bovine Serum Albumin Fraction V, heat shock Millipore-Sigma 3116956001
Chicken anti-GFP Aves GFP-1020
Click-IT plus EdU Alexa Fluor 555 imaging kit, Invitrogen Thermo Fisher Scientific C10638 –
Corn oil Millipore-Sigma C8267 –
Decloaking Chamber Biocare Medical DC2012 –
Dimethyl sulfoxide (DMSO) Fisher BioReagents BP231-100 light sensitive
DNase-free proteinase K Invitrogen C10618H diluted 25x in DPBS
Donkey anti-chicken AF647 Jackson ImmunoResearch 703-605-155
DPBS Fisher Scientific 21-031-CV –
Eosin Fisher Scientific S176
Fluorescence Microscope Nikon Eclipse 90i Bright and fluoerescent light, with objectives: 10X, 20X Nikon
Fluoromount Aqueous Mounting Medium Millipore-Sigma F4680-25ML
Gamma Cell 40 Exactor Best Theratronics Ltd. – 0.759 Gy min-1
Goat anti-rabbit AF488 Jackson ImmunoResearch 111-545-144
Hematoxylin Solution, Gill No. 3 Millipore-Sigma GHS332
HM 325 Rotary Microtome from Thermo Scientific Fisher Scientific 23-900-668
Hoechst 33258, Pentahydrate (bis-Benzimide) Thermo Fisher Scientific H3569 dilution 1:1000
Hydrogen Peroxide Solution, ACS, 29-32%, Spectrum Chemical Fisher Scientific 18-603-252 –
In Situ Cell Death Detection Kit, Fluorescein (Roche) Millipore-Sigma 11684795910
Liquid Blocker Super PAP PEN, Mini Fisher Scientific DAI-PAP-S-M
Lithium Carbonate (Powder/Certified ACS), Fisher Chemical Fisher Scientific L119-500 0.5g/1L dH2O
Luer-Lok Syringe sterile, single use, 10 mL VWR 89215-218 –
Methanol VWR BDH1135-4LP
Pharmco Products Ethyl alcohol, 200 PROOF Fisher Scientific NC1675398 –
Pharmco-Aaper 281000ACSCSLT Acetic Acid ACS Grade Capitol Scientific AAP-281000ACSCSLT –
Rabbit anti-Ki67 BioCare Medical CRM325
Richard-Allan Scientific Cytoseal XYL Mounting Medium Fisher Scientific 22-050-262
Scientific Industries Incubator-Genie for baking slides at 65 degree Fisher Scientific 50-728-103
Sodium Citrate Dihydrate Fisher Scientific S279-500
Stainless Steel Dissecting Kit VWR 25640-002
Superfrost Plus micro slides [size: 25 x 75 x 1 mm] VWR  48311-703
Tamoxifen Millipore-Sigma T5648 30 mg/mL in sterile corn oil, preferably fresh or short-sterm storage in -20°C, light sensitive
Tissue-Tek 24-Slide Holders with Detachable Handle Sakura 4465
Tissue-Tek Accu-Edge Low Profile Blades Sakura 4689
Tissue-Tek Manual Slide Staining Set Sakura 4451
Tissue-Tek Staining Dish, Green with Lid Sakura 4456
Tissue-Tek Staining Dish, White with Lid Sakura 4457
Tween 20 Millipore-Sigma P7949
Unisette Processing Cassettes VWR 87002-292 –
VWR Micro Cover Glasses VWR 48393-081
Xylene Fisher Scientific X5P-1GAL
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Intestinal Epithelial RegenerationIonizing RadiationGamma IrradiationColorectal CancerLineage TracingTamoxifenEdUBouin’s FixativeGamma IrradiatorCre-mediated Recombination
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Orzechowska-Licari, E. J., LaComb, J. F., Giarrizzo, M., Yang, V. W., Bialkowska, A. B. Intestinal Epithelial Regeneration in Response to Ionizing Irradiation. J. Vis. Exp. (185), e64028, doi:10.3791/64028 (2022).
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